Coumarin analog compounds for safer anticoagulant treatment

ABSTRACT

The present invention relates to the albumin-guided development of coumarin analogs and the analogs developed thereby. The coumarin analogs of the present invention are identified and isolated by the fact that they have binding sites to albumin which are different than the binding sites of conventional coumarin analogs such as sodium warfarin, and as a result will be less prone to be displaced since the binding site of sodium warfarin is shared by numerous drugs such as aspirin. The coumarin analogs of the invention are advantageous because they can achieve the effects of the prior coumarin analogs with a minimum of metabolic complications and undesirable side effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/629,858, filed Jul. 30, 2003, which claims the benefit ofU.S. Provisional Application Ser. No. 60/399,126, filed Jul. 30, 2002.

FIELD OF THE INVENTION

The invention relates to the albumin-guided development of certaincoumarin analog compounds which can be used as safer and more effectivealternatives to previous analogs such as Sodium Warfarin, as well asmethods for obtaining the coumarin analogs by assessing and utilizingthe albumin-binding characteristics of the individual compounds so thatthe coumarin analogs of the invention may be identified or designedwhich will bind at sites which are less likely to be displaced by otherdrugs and which will thus result in safer, more controlled levels ofcoumarin analog in the bloodstream. Accordingly, the invention alsorelates to methods of providing anti-coagulant or anti-thrombotictreatment utilizing effective amounts of the coumarin analogs of thepresent invention which will have reduced potential for unfavorable druginteractions, reduced metabolic complications, and improvedcontrollability within their therapeutic range.

BACKGROUND OF THE INVENTION

It has long been known to utilize coumarin analogs for various purposessuch as to provide anti-thrombotic effects where needed. For example,one such analog known as Sodium Warfarin, which has been marketed underthe tradename “Coumadin®” by the DuPont company, is considered thegenerally accepted standard in the field of coumarin analogs, althoughthis drug has been marketed as a generic drug under other brand namessuch as Marevan; Prothromadin; Tintorane; Warfarin sodium; Warfilone;and Waran. Coumarin analogs such as sodium warfarin are disclosed, forexample, in U.S. Pat. Nos. 2,427,578; 2,765,321; 5,591,403; and6,512,005, incorporated herein by reference, and these compounds havebeen utilized primarily for the ability to provide localizedanti-thrombotic effects as needed during therapeutic treatment.

However, such presently available coumarin analogs suffer from variousdrawbacks, including the fact that they may induce undesirable sideeffects. One example of undesirable effects relates to the extensivelisting of unfavorable drug interactions for sodium warfarin, one infact which is the most extensive among approved pharmaceuticals. Many ofthese unfavorable drug interactions arise because these analogs havebeen determined to bind serum albumin at a binding site common to manyother drugs and compounds. This albumin binding site for many drugs isclassically referred to as the Sudlow Site II. As a result, particularlyin cases wherein a patient taking anti-coagulant drugs is taking asecond medication at the same time, there is a great likelihood that thecoumarin analog drug will be displaced from the serum albumin, resultingin the very dangerous situation where the analog will have an increasedactive concentration in the bloodstream which can result in seriousmetabolic consequences and other harmful conditions. Moreover, theuseful life of the coumarin drug can be severely restricted and thus thepatient may not be getting a sufficient supply of the blood-thinningdrug which also may result in severe problems including blood clots,embolism, and even stroke.

Thus, although there have been numerous coumarin analogs synthesized andstudied over the past several decades, none of these have been developedwith a focus on the albumin protein binding characteristics of thevarious coumarin drugs that have been developed, and with the currentheavy emphasis and expense in achieving product safety and efficacy, aswell as regulatory approval, such an approach will be extremely helpfuland cost effective in designing and selecting safe and effectivecoumarin alternatives earlier in the dug development process. It is thusneeded in the field drug develop methods of obtaining coumarinalternatives using a focus which is structurally based on their proteinbinding properties, particularly the binding properties with serumalbumin so as to best determine which analogs will bind to sites whichhave a far lesser likelihood of being displaced by other drugs orparticular conditions in the bloodstream and which provide a stable andcontrolled reservoir of active drug in the blood stream.

SUMMARY OF THE INVENTION

It is thus an object of the present invention to develop coumarinanalogs for use as pharmaceuticals which would directly mimic treatmentsfor which Sodium Warfarin is used, yet which avoid the albumin bindingsites of sodium warfarin so as to be safer and when bound to the albuminat favorable locations provides a stable and more controlled active drugin the bloodstream.

It is further an object of the present invention to utilize the atomicdetermination of serum albumin binding sites in the development of thecoumarin analogs of the present invention, thus providing definitiveinformation on each analog which can bind to alternative sites,producing lead compounds which are less likely to be displaced in thepresence of competing drugs.

It is still further an object of the present invention to developimproved drugs which can be used in place of sodium warfarin withreduced drug interactions, reduced metabolic complications, and improvedcontrollability within therapeutic ranges.

It is even further an object of the present invention to develop methodsfor identifying and isolating albumin-binding coumarin analogs whichbind to alternative sites on the albumin and which are useful intherapeutic treatment methods, and the coumarin analogs identified andisolated thereby.

It is yet further an object of the invention to develop methods foradministering an anti-coagulant agent in amounts effective to achieve adesired therapeutic anti-coagulant or anti-thrombotic effect byadministering an effective amount of the alternative coumarin analogsdeveloped in accordance with the present invention.

These and other objects are achieved by virtue of the present inventionwhich provides novel coumarin analogs useful in treatments whichcurrently use conventional sodium warfarin products such as Coumadin®,along with their enantiomeric forms, derivatives, and active metabolicproducts as well. The coumarin analogs of the present invention areadvantageous in that they are selected by virtue of their affinity forbinding sites on albumin other than the binding site common to sodiumwarfarin and the other forms of warfarin, a site which also forms thebinding site for numerous other drugs and compounds. As a result, thecoumarin analogs of the present invention will bind at sites which havehigh affinity for the analog, yet are far less likely to be displaced byother drugs because the alternative sites do not appear to have a largenumber of competing drugs or compounds that bind at those sites. Thecoumarin analogs of the present invention can thus provide the sameeffective anti-coagulant properties of the prior art warfarin compoundsbut are safer and more effective because of the greater likelihood thatthe analogs of the invention will be in stable equilibrium betweenalbumin and the active free drug in the bloodstream and will not be assusceptible to drug displacement interactions which may result indangerous, higher than desired effective dosing. The present inventionalso provides a method for identifying and isolating these novel andeffective coumarin analogs by virtue of their ability to bind withalternative sites on albumin, or active albumin fragments, and providesmethods of anti-coagulant therapy wherein an effective amount of thecoumarin analog of the present invention is administered to a patient inneed of such therapy.

These and other features of the present invention as set forth in, orwill become obvious from, the detailed description of the preferredembodiments provided hereinbelow.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 is a crystallographic view of the albumin binding properties ofcoumarin analog compound NCP 014 at the binding region IB in accordancewith the present invention.

FIG. 2 is a crystallographic view of the albumin binding properties ofcoumarin analog compound NCP 014 at the binding region IIA in accordancewith the present invention.

FIG. 3 is a crystallographic view of the overall albumin bindingproperties of coumarin analog compound NCP 014 in accordance with thepresent invention.

FIG. 4 a is a crystallographic view of the difference in electrondensity (unbiased and unrefined) of the compound NCP014 in accordancewith the present invention as bound in site IB of serum albumin.

FIG. 4 b is a crystallographic view of the difference in electrondensity (unbiased and unrefined) of the compound NCP014 in accordancewith the present invention as bound in site IA of serum albumin.

FIG. 5 is a crystallographic view of the binding of the prior art sodiumwarfarin at its subsite binding region located in region IIA of serumalbumin which also shows that the binding of coumarin analog NCP 014 inaccordance with the invention binds serum albumin at a separate bindingsite within the binding region IIA.

FIG. 6 a is a crystallographic view of the difference electron density(unbiased and unrefined) of the compound NCP023 in accordance with thepresent invention as bound within its unique single site bindinglocation within the albumin binding region known as subdomain IB.

FIG. 6 b is a crystallographic view of the atomic interactions of NCP023within the IB binding location, also shown together with the presence ofmyristate nearby, but not overlapping with NCP023.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, a system for drug developmentis provided wherein coumarin analogs are identified and isolated byvirtue of their unique and unusual binding sites on serum albuminrelative to other coumarin based anti-coagulants. The term coumarinanalog or coumarin compound refers to those compounds, enantiomers andderivatives of coumarin which are derived from coumarin and retain allof the biological activity of coumarin. By “anti-coagulant” or“anti-coagulant agent” means a coagulation inhibitory agent, or an agentthat treats, inhibits or prevents blood coagulation. Such agents arecommonly known and utilized when therapeutically or medically necessaryand include such agents as warfarin and heparin. Other forms of warfarinanti-coagulant agents include crystalline warfarin, sodium warfarin oramorphous sodium warfarin. These compounds are highly potentanticoagulants and are generally administered orally and usedextensively as active pharmaceutical ingredients (APIs) in amountseffective to achieve the level of anti-coagulation or anti-thrombosis asneeded for a particular patient. Thus, as recognized by one skilled inthe art, the effective amount of any such agent, including agents andcompounds in accordance with the present invention as described furtherbelow, will be that amount which will achieve a desired effect, e.g.,appropriate prevention of blood coagulation, for a particular patientdepending on the size, gender and medical condition of the patient.

In accordance with the present invention, there is provided a method foridentifying and isolating coumarin analogs which have improvedproperties which comprises obtaining candidate coumarin analogs whichretain the anticoagulant properties of coumarin and other analogs suchas sodium warfarin, and introducing said coumarin analog to a serumalbumin, such as human serum albumin or rat serum albumin, such that thebinding characteristics of the candidate coumarin analog can bedetermined, including the specific binding site or subsite therein ofthe coumarin analog to albumin. By “serum albumin” is thus meant anyserum albumin or active fragment thereof which will exhibit binding to acoumarin analog. Previously, conventional coumarin analogs such assodium warfarin suffer from the drawback that they bind to serum albuminat a classical major site (in the IIA region) which is very common andwhich will also constitute the binding site for other drugs andcompounds such as aspirin and numerous other drugs which may beadministered along with an anti-coagulant or anti-thrombotic drug suchas sodium warfarin. The problem is that when the second drug isadministered to the patient, there is a great likelihood of drugdisplacement at the common IIA binding site where sodium warfarin andother similar analogs will bind to the serum albumin. As a result ofthis drug displacement, the prior art coumarin analogs have a fargreater likelihood of being displaced and becoming free in the bloodstream. This possibility presents severe potential damage to the patientin a number of ways. In particular, the elevated amount of free coumarinanalog in the bloodstream can itself lead to a wide variety of metabolicand cardiovascular problems since this drug can have potentially drasticeffects if not properly controlled. Even further, to the extent thecoumarin drug in free form is eliminated more quickly from thebloodstream that would normally occur in the state wherein the analogwas bound to albumin, the patient may not receive the anticoagulanteffect desired by the use of the drug and may thus suffer the conditionsthat are intended to be avoided by the anticoagulant treatment, e.g.,artery blockage, blood clots, embolism and stroke.

To the contrary, the present invention is designed to determine whichcoumarin analogs bind to site on serum albumin which are not the same asthe site targeted by the commonly used coumarin alternatives,particularly sodium warfarin. By determining the albumin bindingcharacteristics of potential candidate coumarin analogs, and thenselecting those analogs which bind at a site other than the site aswhich coumarin analogs such as sodium warfarin will bind, an improvedcoumarin analog will be obtained that will suitably be bound withalbumin at a different location than sodium warfarin, and such acoumarin analog will be far less likely to be displaced by other drugs,endogenous ligands or other conditions in the bloodstream, and willconstitute a far safer and more effective compound for anti-coagulanttreatment. Accordingly, in accordance with the present invention, animproved coumarin analog is obtained which will bind to an albuminbinding site or region which will have altered affinity for the coumarinanalog and which will be much less likely to be subject to drugdisplacement. The resulting coumarin analogs in accordance with thepresent invention will thus be more controllable than prior art coumarinalternatives, and can also be used with greater safely and efficacy withfewer side effects such as metabolic complications.

In accordance with the present invention, there are provided coumarinanalog compounds, the parent compounds for which do not bind at the samealbumin binding site as prior conventional coumarin analogs such assodium warfarin. In particular, the following specific coumarin analogcompounds, identified herein as NCP 014, 014a, 014b, 023, and 023a-h,have been identified, isolated or suggested in accordance with thepresent invention, and such compounds will maintain the ability to actas anticoagulant agents, but will not be subject to drug displacement inthe same manner as conventional coumarin analogs such as sodium warfarinwhich bind to the subsite located in the IIA binding region of albumin:

NCP023, NCP023a, and NCP014, among many other coumarin analogs, wereexamined in accordance with the present invention to determine thenature of the binding of albumin so as to identify and isolate thoseanalogs which will have a binding site different from that of sodiumwarfarin and which will thus be suitable for use in more stable andsafer anticoagulant therapy in accordance with the present invention.Crystallographic information reflecting these analogs is shown in FIGS.1-3,4 a and 4 b, 5, and 6 a and 6 b in accordance with the inventionwherein coumarin analogs NCP 014 and NCP023 are shown to bind to bindingregion IB and/or IIA binding regions of human serum albumin, but at asite which differs from the major, well known site of sodium warfarin inthe IIA region. The crystallographic pictures showing the binding ofsodium warfarin inside the IIA binding region of serum albumin is shownin FIG. 5. As also shown in FIG. 5, the warfarin/Coumadin® binding siteis distinct from the binding location of NCP 014 within the albumin IIAbinding region.

The above coumarin analogs in accordance with the present invention willthus be suitable for use in anticoagulant therapy and will provide amore stable reservoir of the anticoagulant drug than in cases whereinanalogs such as sodium warfarin are used which are more subject to drugdisplacement. In the preferred embodiment, the coumarin analog compoundsof the present invention will be utilized in composition wherein asuitable and pharmaceutically acceptable vehicle, carrier or excipientis used as would be recognized by one of ordinary skill in this art. Thevehicle, excipient or carrier may be any suitable pharmaceuticalvehicle, excipient or carrier that would commonly be used in this art,including such as saline, dextrose, water, glycerol, ethanol, othertherapeutic compounds, and combinations thereof. As one skilled in thisart would recognize, the particular vehicle, excipient or carrier usedwill vary depending on the patient and the patient's condition, and avariety of modes of administration would be suitable for thecompositions of the invention, as would be recognized by one of ordinaryskill in this art. Generally, the compositions of the present inventionare designed for oral administration, but alternative methods ofadministration when necessary in particular cases include anal, vaginal,intravenous, intraperitoneal, intramuscular, subcutaneous, intranasaland intradermal when oral administration is not feasible.

In addition, the coumarin analog compounds of the invention may beemployed in anticoagulant compositions wherein the compounds areincluded in an amount effective to achieve the desired anticoagulanteffect, e.g., the prevention of blood coagulation, in the patient. Asindicated above, by “effective amount” is meant that nontoxic butsufficient amount of the anticoagulant agent which will obtain thedesired inhibitory, prophylactic or therapeutic effect in the patient inneed of anticoagulant therapy. As will be pointed out below, the exactamount of the antibody or a particular agent that is required will varyfrom subject to subject, depending on the species, age, gender andgeneral condition of the subject, the severity of the condition beingtreated, the particular carrier or adjuvant being used and its mode ofadministration, and the like. Accordingly, the “effective amount” of theactive agent or compositions containing the active agent will vary basedon the particular circumstances, and an appropriate effective amount maybe determined in each case of application by one of ordinary skill inthe art using only routine experimentation. The dose should be adjustedto suit the individual to whom the composition is administered and willvary with age, gender, weight and metabolism of the individual. Thecompositions may additionally contain stabilizers or pharmaceuticallyacceptable preservatives, such as thimerosal(ethyl(2-mercaptobenzoate-S)mercury sodium salt) (Sigma ChemicalCompany, St. Louis, Mo.).

In additional embodiments, the coumarin analogs of the present inventionmay be utilized in mixtures wherein additional anticoagulant compoundssuch as sodium warfarin may be used. Such compounds may form a combinedtreatment regimen wherein the effect of the initial anticoagulant basedon sodium warfarin is immediate but not continued long-term, and theeffect of the coumarin analog of the present invention, as controlled byits ability to be retained by serum albumin, will give a farlonger-lasting anticoagulant effect.

In short, the coumarin analogs of the present invention are advantageousin that they are selected by virtue of their affinity for binding siteson albumin other than the binding site common to sodium warfarin and theother forms of warfarin, a site which also forms the binding site fornumerous other drugs and compounds, and compositions made which includethese compounds will be advantageous in their effectiveness foranticoagulant therapy without harmful side effects. The coumarin analogsof the present invention will thus bind at sites which have alteredaffinity for the analog, yet are far less likely to be displaced byother drugs because the alternative sites do not appear to have a largenumber of competing drugs or compounds that bind at those sites. Thecoumarin analogs of the present invention can thus provide the sameeffective anti-coagulant properties of the prior art warfarin compoundsbut are safer and more effective because of the greater likelihood thatthe analogs of the invention will remain in a more controlledequilibrium with the serum albumin in the bloodstream and not bereleased from albumin at undesirable levels due to drug or endogenousligand displacement interactions.

In another aspect of the present invention, the above described coumarincompounds can be utilized in methods of anti-coagulant therapy whereinan effective amount of the coumarin analog of the present invention isadministered to a patient in need of such therapy. As indicated above,by “effective amount” is meant that amount that will be administered toa given human or animal patient so as to achieve an a desiredanticoagulant result, e.g., the prevention of blood coagulation when sodesired in a patient in need of therapy, and such an amount will bedependent on a variety of factors such as patient gender, size andcondition and will thus vary from patient to patient as would berecognized by one of ordinary skill in the art. Accordingly, methods areprovided wherein treatment for problems associated with excessive bloodcoagulation, e.g., stroke and embolism, is accomplished by administeringan effective amount of the coumarin analogs of the present invention, orcompositions containing said coumarin analogs as described in detailabove.

Another aspect of the present invention is thus a method for achievingsafe and effective blood coagulation prevention which comprisesadministering to a patient in need of such therapy an effective amountof a coumarin analog in accordance with the invention as describedabove, or a pharmaceutical composition containing said coumarin analogas described above.

In summary, the present invention provides a new method of obtainingsafer and more effective coumarin analogs which can provideanticoagulant or antithrombotic properties and yet not be subject todrug displacement as would be the case with conventional coumarinanalogs such as sodium warfarin which are more likely to be displacedand cause a variety of metabolic and/or medical problems.

It is thus submitted that the foregoing embodiments are onlyillustrative of the claimed invention and not limiting of the inventionin any way, and alternative embodiments that would be obvious to oneskilled in the art not specifically set forth above also fall within thescope of the claims.

The following examples are presently only as illustrative of certainaspects of the present invention and do not act to limit the scope ofthe invention in any way.

EXAMPLES

In the following examples, Biacore and Animal Study data were obtainedwith regard to some of the compounds of the present invention, namelyNCP 014, 023, 023a.

In each case, the studies showed that the particular coumarin analog ofthe invention bound to albumin at a binding site distinct from that ofsodium warfarin.

Example 1 BIACORE and Animal Study Data on NCP 014

NCP Protein Binding Sites K_(D1) μM K_(D2) μM 014 HSA 2 0.7 130 014 HSA2 0.8 110 014 HSA 2 4.6 Not provided 014 HSA 1 6.5 N/A 014 HSA Fragment2 0.3 97 014 HSA Fragment Not determined Not determined Not determined014 HSA Fragment 2 18.0 Not provided 014 HSA Fragment 1 63.0 N/A 014 RatAlbumin 2 4.6 Not providedProthrombin Time (PT) ˜52 sec, 24 hrs after 25 mg bolus of NCP 014delivered orally in water

In this case, NCP 014 was observed to bind at the IB binding site, andin addition in the IIA binding site, but at a location different thanthe major site wherein sodium warfarin binds to albumin.

Example 2 BIACORE and Animal Study Data on NCP 023 and 023a

NCP Protein Binding Sites K_(D1) μM K_(D2) μM 023 HSA 2 1.7 112 023 HSA2 2.4 160 023 HSA 2 20.0 Not provided 023 HSA 1 103.0 N/A 023 HSAFragment 2 2.7 220 023 HSA Fragment 1 83.0 N/A 023 HSA Fragment Notdetermined Not determined Not determined 023 Rat Albumin 85.0 Notprovided Prothrombin Time (PT) ˜50 sec, 24 hrs after 25 mg bolus of NCP023 delivered orally in water 023a HSA 1 23.0 N/A 023a HSA 2 0.5 40 023aHSA 2 12.0 Not provided 023a HSA 1 66.8 N/A 023a HSA Fragment 2 0.7 50023a HSA Fragment Not determined Not determined Not determined 023a HSAFragment 1 79.0 N/A 023a HSA Fragment 1 122.0 N/A 023a Rat Albumin 211.8 Not provided Prothrombin Time (PT) ˜61 sec, 24 hrs after 25 mgbolus of NCP 023a delivered orally in water

In these cases, NCP 023 and 023a were observed to bind at a site otherthan the IIA binding site at which sodium warfarin binds to albumin. Inparticular, these compounds were determined to bind at the IB site ofserum albumin unique from the IB endogenous long-chain fatty acid site

1. A method of selecting coumarin analogs for effective anticoagulationtherapy with a reduced risk of adverse side effects comprising obtainingcandidate coumarin analogs having anticoagulation activity, introducingsaid candidate analogs to serum albumin in such a manner so that thebinding location of the candidate coumarin analog to serum albumin canbe determined, determining the location of the binding site of saidcandidate analog to serum albumin, and selecting said coumarin analogswhich bind to serum albumin at a binding site different from that ofsodium warfarin.
 2. A coumarin analog obtained by the method of claim 1.3. An anticoagulant composition comprising the coumarin analog of claim2 and a pharmaceutically acceptable vehicle, carrier or excipient.
 4. Ananticoagulant composition comprising a coumarin analog having theformula:

and a pharmaceutically acceptable vehicle, carrier or excipient.
 5. Amethod of inhibiting or preventing blood coagulation in a patient inneed of such therapy comprising administering to said patient thecomposition according to claim 4 in an amount effective to achieveinhibition or prevention of blood coagulation.
 6. A method of inhibitingor preventing blood coagulation in a patient in need of such therapycomprising administering to said patient a coumarin analog having theformula:

in an amount effective to achieve inhibition or prevention of bloodcoagulation.